The present invention relates to antibodies and antibody fragments, and methods of making and using these antibodies and antibody fragments, in the diagnosis, prognosis, prevention and treatment of diseases. The antibodies and antibody fragments are capable of binding to soluble and membrane bound EGFR, as well as to phosphorylated EGFR with high specificity, high sensitivity, and high affinity, and are thus useful in both therapeutic and diagnostic applications.
The epidermal growth factor receptor (EGFR; ErbB; ErbB-1; HER1), a 170 kDa transmembrane glycoprotein encoded by the human HER1 gene, is a member of the protein kinase superfamily. EGFR is a cell-surface receptor that is a member of the epidermal growth factor family (EGF-family). It consists of an extracellular ligand binding domain, a transmembrane domain and an intracellular domain with intrinsic protein-tyrosine kinase activity. See Modjtahedi et al. (1996) Br. J. Cancer 73: 228-235; Herbst and Shin (2002) Cancer 94: 1593-1611. The soluble form of EGFR is secreted from cells and can be recognized using the anti-EGFR antibodies disclosed herein. See Weber and Gill (1984) Science 224: 294-297.
EGFR binds to different ligands, including, but not limited to, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), amphiregulin, heparin-binding EGF (hb-EGF), betacellulin, and epiregulin. See Herbst and Shin (2002) Cancer 94: 1593-1611; Mendelsohn and Baselga (2000) Oncogene 19: 6550-65.
EGFR has been associated with many pathological disorders. For example, overexpression of EGFR has been reported in numerous human malignant conditions, including cancers of the bladder, brain, head and neck, pancreas, lung, including non-small cell lung cancer, breast, ovary, colon, prostate, and kidney. See Atalay et al. (2003) Ann. Oncology 14: 1346-1363; Herbst and Shin (2002) Cancer 94: 1593-1611; Modjtahedi et al. (1996) Br. J. Cancer 73: 228-235. In many of these conditions, the overexpression of EGFR correlates or is associated with poor prognosis of the patients. Id.
EGFR is abnormally activated (e.g., overexpressed, mutated) in many epithelial tumors, including non-small cell lung cancer, breast cancer, colorectal cancer, head and neck cancers, and prostate cancer. See Adams, G. and Weiner, L. (2005) Nature Biotechnology, 23: 1147-1157. Abnormal activation of EGFR can arise from overexpression of the receptor, gene amplification, activating mutations, overexpression of receptor ligands, and/or loss of regulators of EGFR activity. See Baselga, J. and Arteaga (2005) J. of Clin. Oncol. 23: 2445-2459. Abnormally high EGFR activation results in phosphorylation of several intracellular substrates, which in turns gives rise to mitogenic signaling as well as other tumor-inducing activities. Consequently, EGFR is a target for anti-cancer therapeutic strategies which can potentially inhibit or reduce the receptor's aberrant expression, and/or its activation.
Anti-cancer agents that target EGFR include monoclonal antibodies. The chimeric monoclonal antibody C225 (or cetuximab), which contains the murine variable region of mAb225 and a human IgG1 constant region, is presently available for treatment of certain types of colon cancer in at least the United States and Europe. See Baselga, J. and Arteaga, C. (2005) J. of Clin. Oncol. 23: 2445-2459. The fully human antibody ABX-EGF (panitumumab) is also approved in at least the United States, Europe, and Canada for the treatment of metastatic colorectal cancer. Panitumumab has been reported to have an affinity for EGFR approximately 8-fold greater than that of C225. See Yang, X-D et al. (2001) Crit. Rev. Oncol./Hemat. 38: 17-23. Another humanized anti-EGFR monoclonal antibody, EMD72000 (matuzumab), was in phase II clinical trials in the United States for the treatment of colorectal, lung, esophageal and stomach cancers. See Vanhoefer, U. et al. (2004) J. Clin Oncol. 22: 175-184. It has also been reported that the affinity of humanized antibody h-R3 for EGFR is less than that of C225. See Crombet, T. et al. (2004) J. Clin. Oncol. 22: 1646-1654. Complications, such as skin toxicity that results in flushing, seborrheic dermatitis, and acneform rash have been observed in the clinic with doses of C225 higher than 100 mg/m2. See Herbst, R. and Langer, C. (2002) Semin. Oncol. 29: 27-36. Thus, there is a need for anti-EGFR antibodies that are highly sensitive, highly specific and have high affinity to EGFR for therapeutic purposes.
Anti-EGFR antibodies are also used diagnostically. For example, an anti-EGFR antibody contained in the “EGFR pharmDX™” kit from Dako is FDA-approved for identifying patients with colorectal cancer eligible for treatment with cetuximab or panitumumab. However, there is no evidence that the antibody in this kit recognizes soluble EGFR. Thus, the kit is useful in immunohistochemical (IHC) assays, but would not be useful in assays biological fluids such as blood, serum, urine or plasma.
Available diagnostic anti-EGFR antibodies have limited use since they do not recognize soluble EGFR such as would be found in the blood, serum, plasma, or urine. There is a need for anti-EGFR antibodies that are highly sensitive, highly specific and have high affinity to soluble and membrane bound EGFR for diagnostic purposes. There is also a need for anti-EGFR antibodies that are able to recognize the soluble and phosphorylated form of EGFR so that assays can be conducted on blood, plasma, serum, and urine samples rather than tissue biopsies. Less invasive procedures for detecting aberrant EGFR expression, and thus diagnosing EGFR-related diseases and conditions could be accomplished with such an antibody.
The invention is based in part on the creation of an anti-EGFR monoclonal antibody with superior and unexpected beneficial properties, including, but not limited to: a capability to bind to the extracellular domain of EGFR; a capability to bind to the soluble form of EGFR; a capability to bind to a phosphorylated form of the antibody; a superior binding affinity to EGFR; an extra-high selectivity and specificity to EGFR; improved blocking properties; extra-low immunogenicity; extra low toxicity; increased utility for targeting EGFR-expressing cells; increased Fc receptor binding and increased effector function.
Additional objects and advantages of the invention will be set forth in part in the description which follows. The objects and advantages of the present invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention, as claimed.